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Plant Cell. Nov 1998; 10(11): 1927–1940.
PMCID: PMC143949

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells


Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.

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Selected References

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  • Asada T, Shibaoka H. Isolation of polypeptides with microtubule-translocating activity from phragmoplasts of tobacco BY-2 cells. J Cell Sci. 1994 Aug;107(Pt 8):2249–2257. [PubMed]
  • Baas PW, Brown A. Slow axonal transport: the polymer transport model. Trends Cell Biol. 1997 Oct;7(10):380–384. [PubMed]
  • Baskin TI, Wilson JE. Inhibitors of protein kinases and phosphatases alter root morphology and disorganize cortical microtubules. Plant Physiol. 1997 Feb;113(2):493–502. [PMC free article] [PubMed]
  • Blackman LM, Boevink P, Cruz SS, Palukaitis P, Oparka KJ. The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii . Plant Cell. 1998 Apr;10(4):525–538. [PMC free article] [PubMed]
  • Blancaflor EB, Hasenstein KH. Growth and microtubule orientation of Zea mays roots subjected to osmotic stress. Int J Plant Sci. 1995 Nov;156(6):774–783. [PubMed]
  • Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802–805. [PubMed]
  • Chan J, Rutten T, Lloyd C. Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus. Plant J. 1996 Aug;10(2):251–259. [PubMed]
  • Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J. Engineered GFP as a vital reporter in plants. Curr Biol. 1996 Mar 1;6(3):325–330. [PubMed]
  • Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY. Understanding, improving and using green fluorescent proteins. Trends Biochem Sci. 1995 Nov;20(11):448–455. [PubMed]
  • Cyr RJ. Microtubules in plant morphogenesis: role of the cortical array. Annu Rev Cell Biol. 1994;10:153–180. [PubMed]
  • Cyr RJ, Palevitz BA. Organization of cortical microtubules in plant cells. Curr Opin Cell Biol. 1995 Feb;7(1):65–71. [PubMed]
  • Desai A, Mitchison TJ. Microtubule polymerization dynamics. Annu Rev Cell Dev Biol. 1997;13:83–117. [PubMed]
  • Durso NA, Cyr RJ. A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-1 alpha. Plant Cell. 1994 Jun;6(6):893–905. [PMC free article] [PubMed]
  • Epel BL, Padgett HS, Heinlein M, Beachy RN. Plant virus movement protein dynamics probed with a GFP-protein fusion. Gene. 1996;173(1 Spec No):75–79. [PubMed]
  • Fisher DD, Cyr RJ. Extending the Microtubule/Microfibril paradigm. Cellulose synthesis is required for normal cortical microtubule alignment in elongating cells . Plant Physiol. 1998 Mar;116(3):1043–1051. [PMC free article] [PubMed]
  • Fukuda H. Tracheary Element Differentiation. Plant Cell. 1997 Jul;9(7):1147–1156. [PMC free article] [PubMed]
  • Gu X, Verma DP. Dynamics of phragmoplastin in living cells during cell plate formation and uncoupling of cell elongation from the plane of cell division. Plant Cell. 1997 Feb;9(2):157–169. [PMC free article] [PubMed]
  • Haseloff J, Amos B. GFP in plants. Trends Genet. 1995 Aug;11(8):328–329. [PubMed]
  • Heim R, Tsien RY. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol. 1996 Feb 1;6(2):178–182. [PubMed]
  • Heinlein M, Epel BL, Padgett HS, Beachy RN. Interaction of tobamovirus movement proteins with the plant cytoskeleton. Science. 1995 Dec 22;270(5244):1983–1985. [PubMed]
  • Hepler PK, Hush JM. Behavior of Microtubules in Living Plant Cells. Plant Physiol. 1996 Oct;112(2):455–461. [PMC free article] [PubMed]
  • Hirokawa N. Microtubule organization and dynamics dependent on microtubule-associated proteins. Curr Opin Cell Biol. 1994 Feb;6(1):74–81. [PubMed]
  • Hugdahl JD, Bokros CL, Hanesworth VR, Aalund GR, Morejohn LC. Unique functional characteristics of the polymerization and MAP binding regulatory domains of plant tubulin. Plant Cell. 1993 Sep;5(9):1063–1080. [PMC free article] [PubMed]
  • Hush JM, Wadsworth P, Callaham DA, Hepler PK. Quantification of microtubule dynamics in living plant cells using fluorescence redistribution after photobleaching. J Cell Sci. 1994 Apr;107(Pt 4):775–784. [PubMed]
  • Hush J, Wu L, John PC, Hepler LH, Hepler PK. Plant mitosis promoting factor disassembles the microtubule preprophase band and accelerates prophase progression in Tradescantia. Cell Biol Int. 1996 Apr;20(4):275–287. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lloyd C. Why should stationary plant cells have such dynamic microtubules? Mol Biol Cell. 1994 Dec;5(12):1277–1280. [PMC free article] [PubMed]
  • Ludin B, Matus A. GFP illuminates the cytoskeleton. Trends Cell Biol. 1998 Feb;8(2):72–77. [PubMed]
  • MacRae TH. Microtubule organization by cross-linking and bundling proteins. Biochim Biophys Acta. 1992 Nov 20;1160(2):145–155. [PubMed]
  • Mandelkow E, Mandelkow EM. Microtubules and microtubule-associated proteins. Curr Opin Cell Biol. 1995 Feb;7(1):72–81. [PubMed]
  • Marc J, Sharkey DE, Durso NA, Zhang M, Cyr RJ. Isolation of a 90-kD Microtubule-Associated Protein from Tobacco Membranes. Plant Cell. 1996 Nov;8(11):2127–2138. [PMC free article] [PubMed]
  • McClinton RS, Sung ZR. Organization of cortical microtubules at the plasma membrane in Arabidopsis. Planta. 1997;201(3):252–260. [PubMed]
  • Mitchison T, Kirschner M. Dynamic instability of microtubule growth. Nature. 1984 Nov 15;312(5991):237–242. [PubMed]
  • Moore RC, Zhang M, Cassimeris L, Cyr RJ. In vitro assembled plant microtubules exhibit a high state of dynamic instability. Cell Motil Cytoskeleton. 1997;38(3):278–286. [PubMed]
  • Nogales E, Wolf SG, Downing KH. Structure of the alpha beta tubulin dimer by electron crystallography. Nature. 1998 Jan 8;391(6663):199–203. [PubMed]
  • Olson KR, McIntosh JR, Olmsted JB. Analysis of MAP 4 function in living cells using green fluorescent protein (GFP) chimeras. J Cell Biol. 1995 Aug;130(3):639–650. [PMC free article] [PubMed]
  • Ormö M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ. Crystal structure of the Aequorea victoria green fluorescent protein. Science. 1996 Sep 6;273(5280):1392–1395. [PubMed]
  • Preuss U, Döring F, Illenberger S, Mandelkow EM. Cell cycle-dependent phosphorylation and microtubule binding of tau protein stably transfected into Chinese hamster ovary cells. Mol Biol Cell. 1995 Oct;6(10):1397–1410. [PMC free article] [PubMed]
  • Rizzuto R, Brini M, De Giorgi F, Rossi R, Heim R, Tsien RY, Pozzan T. Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo. Curr Biol. 1996 Feb 1;6(2):183–188. [PubMed]
  • Rodionov VI, Borisy GG. Microtubule treadmilling in vivo. Science. 1997 Jan 10;275(5297):215–218. [PubMed]
  • Sheen J, Hwang S, Niwa Y, Kobayashi H, Galbraith DW. Green-fluorescent protein as a new vital marker in plant cells. Plant J. 1995 Nov;8(5):777–784. [PubMed]
  • Shelden E, Wadsworth P. Stimulation of microtubule dynamic turnover in living cells treated with okadaic acid. Cell Motil Cytoskeleton. 1996;35(1):24–34. [PubMed]
  • Stearns T. Green fluorescent protein. The green revolution. Curr Biol. 1995 Mar 1;5(3):262–264. [PubMed]
  • Takesue K, Shibaoka H. The cyclic reorientation of cortical microtubules in epidermal cells of azuki bean epicotyls: the role of actin filaments in the progression of the cycle. Planta. 1998 Aug;205(4):539–546. [PubMed]
  • van de Sande K, Pawlowski K, Czaja I, Wieneke U, Schell J, Schmidt J, Walden R, Matvienko M, Wellink J, van Kammen A, et al. Modification of phytohormone response by a peptide encoded by ENOD40 of legumes and a nonlegume. Science. 1996 Jul 19;273(5273):370–373. [PubMed]
  • Vaughn KC, Harper JD. Microtubule-organizing centers and nucleating sites in land plants. Int Rev Cytol. 1998;181:75–149. [PubMed]
  • West RR, Tenbarge KM, Olmsted JB. A model for microtubule-associated protein 4 structure. Domains defined by comparisons of human, mouse, and bovine sequences. J Biol Chem. 1991 Nov 15;266(32):21886–21896. [PubMed]
  • Wymer CL, Fisher DD, Moore RC, Cyr RJ. Elucidating the mechanism of cortical microtubule reorientation in plant cells. Cell Motil Cytoskeleton. 1996;35(2):162–173. [PubMed]
  • Wymer CL, Wymer SA, Cosgrove DJ, Cyr RJ. Plant cell growth responds to external forces and the response requires intact microtubules. Plant Physiol. 1996 Feb;110(2):425–430. [PMC free article] [PubMed]
  • Yuan M, Shaw PJ, Warn RM, Lloyd CW. Dynamic reorientation of cortical microtubules, from transverse to longitudinal, in living plant cells. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6050–6053. [PMC free article] [PubMed]
  • Yuan M, Warn RM, Shaw PJ, Lloyd CW. Dynamic microtubules under the radial and outer tangential walls of microinjected pea epidermal cells observed by computer reconstruction. Plant J. 1995 Jan;7(1):17–23. [PubMed]
  • Zandomeni K, Schopfer P. Mechanosensory microtubule reorientation in the epidermis of maize coleoptiles subjected to bending stress. Protoplasma. 1994;182(3-4):96–101. [PubMed]

Articles from The Plant Cell are provided here courtesy of American Society of Plant Biologists


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